During the reading of the genetic code, bacteria use more than 50 different aminoacyl-tRNAs as interchangeable substrates of the ribosome. The long-term goal of this project is to understand why each bacterial tRNA species has a unique, phylogenetically conserved set of nucleotides and post-transcriptional modifications. The project will test the hypothesis that most of this structural diversity has evolved to idiosyncratically tune each tRNA species such the ribosome can decode the different codons with similar rates and minimize misreading of incorrect codons. In other words, individual tRNAs are different so that they can be equivalent substrates of the ribosome and perform accurately in translation. The project will use a combination of biochemistry, biophysics, and molecular genetics to identify the tRNA sequences and post-transcriptional modifications that are responsible for tuning and then understand their mechanism. Several experiments will exploit our recent discovery that the three base pairs in T-stem of tRNA that ensure uniform binding to elongation factor Tu also affect the release of aminoacyl-tRNA from elongation factor Tu on the ribosome. Three different E. coli tRNAs have been chosen for a large scale mutagenic screen for sequence elements that tune tRNAs in decoding by the ribosome. Finally, five poorly studied modifications present in the body of tRNA will be examined for their role(s) in ribosomal decoding. Taken together, these experiments should for the first time provide a comprehensive understanding of how different tRNAs adapt to the ribosome.